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Determining the secondary structure and orientation of EmrE, a multi-drug transporter, indicates a transmembrane
I T Arkin1, W P Russ, M Lebendiker
1Howard Hughes Medical Institute, Yale University, New Haven, Connecticut 06520, USA.
Abstract:
EmrE is a member of a newly emerging family of MiniTEXANS, a family of multi-drug antiporters from bacteria characterized by their small size of roughly 100 amino acids. In this report we have obtained transmission FTIR spectra of EmrE in CHCl3:MeOH, DMPC vesicles, and Escherichia coli lipid vesicles. Secondary structure analysis has shown that both in DMPC vesicles and in CHCl3: MeOH the protein adopts a highly helical secondary structure that correlates remarkably well with that predicted by hydropathy analysis. The protein was shown to be resistant to amide proton H/D exchange, providing evidence that most of the protein is embedded in the lipid bilayer. Polarized ATR-FTIR spectra of the protein in DMPC vesicles have shown that the helices are oriented with an average tilt angle of 27 degrees from the bilayer normal. The protein was found to be less oriented in E. coli lipid vesicles, most likely as a result of the poor orientation of the bilayer lipids themselves. Thus, the protein is identified as a transmembrane four-helix bundle providing valuable structural data for this family of multi-drug transporters. The results set the stage for further studies aimed at deriving a detailed model for this protein.
Insights
EmrE, a bacterial multi-drug transporter, is a transmembrane four-helix bundle. FTIR studies reveal its helical structure and orientation within lipid bilayers, crucial for understanding drug transport mechanisms.
Area of Science:
- Biochemistry
- Structural Biology
- Membrane Proteins
Background:
- EmrE is a small (approx. 100 amino acids) multi-drug antiporter belonging to the MiniTEXANS family.
- MiniTEXANS are an emerging class of bacterial transporters with significant implications for drug resistance.
Purpose of the Study:
- To determine the secondary and tertiary structure of the EmrE protein.
- To investigate the orientation and membrane integration of EmrE within lipid bilayers.
- To provide structural insights into the MiniTEXANS family of multi-drug transporters.
Main Methods:
- Transmission FTIR spectroscopy of EmrE in solution (CHCl3:MeOH) and lipid vesicles (DMPC and E. coli).
- Amide proton hydrogen-deuterium (H/D) exchange to assess membrane embedding.
- Polarized Attenuated Total Reflection FTIR (ATR-FTIR) to determine helix orientation.
Main Results:
- EmrE exhibits a highly helical secondary structure in both solution and lipid bilayers, consistent with hydropathy predictions.
- The protein is resistant to H/D exchange, indicating extensive embedding within the lipid bilayer.
- Polarized ATR-FTIR revealed an average helix tilt angle of 27 degrees from the bilayer normal in DMPC vesicles.
Conclusions:
- EmrE is confirmed as a transmembrane four-helix bundle.
- Structural data provides valuable insights into the mechanism of this multi-drug transporter family.
- The findings lay the groundwork for detailed structural modeling of EmrE and related transporters.